Summary: Overview: This research program addresses basic molecular and physiological processes of nociceptive transmission in the central and peripheral nervous systems and new ways to effectively control pain. The molecular research is performed using animal and in vitro, cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are investigated through examination of events and molecules in damaged or inflamed peripheral tissue in primary cultures or using reductionist approaches heterologous expression systems. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) to investigate mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new analgesic treatments for severe pain. The current approach, based on our studies of pain transduction through the vanilloid receptor 1 ion channel, now called TRPV1, has resulted in a clinical trial of resiniferatoxin (RTX) as a new treatment for advanced cancer pain. RTX activates TRPV1, which is a heat-sensitive calcium/sodium ion channel that normally converts painful heat into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and triggers action potentials that are conducted to the spinal cord. RTX is a potent vanilloid agonist (capsaicin-like molecule) that props open the TRPV1 ion channel, causing calcium cytotoxicity and death of TRPV1 pain-sensing neurons. This produces very effective pain control in pre-clinical models by several routes of administration: exposure of the cell body leads to cell death (e.g. intraganglionic injections), peripheral application to nerve endings in skin and joints produces a temporary effect. Through an inter-institute working group established with NIDA's Division of Pharmacotherapies and Medical Consequences of Drug Abuse, we finalized an IND application and submitted it to the FDA late in 2008. We also submitted the finalized clinical protocol to the NCI IRB;approval of both was obtained in late 2008. We are now conducting the first human clinical trial using RTX in cancer pain. <http://clinicalstudies.info.nih.gov/detail/A_2009-D-0039.html>. This is an open-label trial to determine the safety of RTX administered into the CSF surrounding the spinal cord (intrathecal). Patient recruitment began January, 2009;the first patient was treated in September, 2009. This patient had intractable pain from advanced cervical cancer. The treatment completely eliminated her cancer pain with no adverse side effects on motor or somatosensorry functions. Removal of pain produced mood elevation, increased activity, and increased appetite. Our plan is to complete the Phase I study, generate a new protocol for PHN patients and establish protocols for controlling other acute and chronic pain conditions such as joint pain, head and neck cancer, and neuropathic pain. Basic Pain Mechanisms: Underlying the translational studies are investigations of molecular regulation of gene expression, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. The goal of this approach is to obtain a comprehensive and informative understanding of nociceptive process. The dorsal root ganglion is quite small but its analysis is made possible by the extensive use of reverse-transcription-PCR, which allows us to make multiple measurements on such small tissue samples. Our studies reveal a complex, dynamic modulation of gene expression at all three steps. We have examined novel molecules as well as neuropeptide, cytokine and chemokine expression and identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. The functional implications of our studies on cytokines suggest a roles for monocyte chemoattractant protein 1 in nociceptive DRG neurons and choroid plexus in inflammatory states. This molecule is enriched in dorsal spinal cord where it mediates intercellular communication. Further studies demonstrated a highly enriched eexpression in the choroid plexus where it may have a more broad action on the entire nervous system by influencing composition of the cerebrospinal fluid. The above studies were conducted in the laboratory rat, we are now examining human DRG and spinal cord to determine if the same expression and enrichment occurs in human pain circuits. We established collaboration with the Neuropathology Section of the Clinical Brain Disorders Branch of NIMH to collect human trigeminal ganglia and the spinal trigeminal nucleus when tissue is obtained for their brain bank. These tissues shall be used for analysis of peptides, proteins and expression profiles to bridge the gap between rodent and human nociceptive circuits. Through this research in animals and humans we hope to obtain a fundamental understanding of the relationships between tissue damage, inflammation and pain sensation. In a broader framework, these studies explore the fundamental molecular basis of synaptic plasticity. We hypothesize modularity in neuronal responses when a new level of synaptic or pharmacological input occurs that will be relevant not only to pain, but also to situations such as learning, neurological disorders like epilepsy, and drug abuse. A set of "generic" alterations is combined with circuit-specific genes to meet the demands of new stimulation or activity. Understanding the molecular repertoire and its dynamic interactions will lead to a deeper understanding of mechanisms that trigger and sustain chronic pain and other disorders of the nervous system. Early Translational Investigations: A final set of studies concerns the identification of small chemicals that act as allosteric potentiators of vanilloid agonists on TRPV1. Our cancer study shows that this ligand-gated ion channel is one of the most important molecular transducers of painful stimuli and understanding how TRPV1 can be blocked, activated or sensitized is of primary importance in understanding pain. We have identified a new action on TRPV1 via screening of a targeted calcium channel chemical library. This activity is manifested as an enhancement of activation of TRPV1 calcium influx by agonist binding to the orthosteric site. These studies suggest an allosteric modulation of the open state of the TRPV1 ion channel and the presence of a reserve level of activity that can be accessed for pain transmission as well as the existence of a new class of pharmacological agents for pain modulation and pain control. Our initial studies focused on the identification of this class of modulators. We have since extended our calcium-45 uptake studies with live cell imaging observation using manipulations of calcium concentrations, electrophysiological recordings from heterologous TRPV1-expressing cell lines and examination of other modalities of TRPV1 stimulation (e.g. heat, pH) to determine if they are enhanced or modulated by the allosteric potentiators.